Type I interferons (IFN-a/b, hereafter referred to as IFN) are a family of cytokines necessary for the stimulation of effective anti-viral host defense. Both Toll Like Receptor (TLR) dependent and TLR-independent mechanisms exist for the production of type I IFNs following viral infection1. In an emerging picture of TLR- independent responses to viruses, dsRNA or uncapped ssRNA species produced during the course of virus infection (but not normally found in an uninfected cell) are recognized by the intracellular helicases melanoma differentiation antigen (MDA)-5 and retinoic acid inducible gene (RIG)-I, respectively. This event activates production of IFN via a mitochondrial protein called interferon promoter stimulator (IPS)-1 to activate IFN production1. Importantly, our data and those of others show that the death-domain (DD)-containing adaptor proteins FADD (Fas-asscociated protein with death domain) and RIP1 (receptor interacting protein kinase 1) are also essential for optimal signaling by MDA-5, RIG-I and IPS-1. Although essential roles have been established for RIG-I, MDA-5, IPS-1, FADD and RIP1 in innate immune responses to virus infection through the use of homozygous-null mice and cells, the manner in which these key molecules are activated by virus infection to stimulate IFN gene transcription are poorly defined. Indeed, as our data below indicate, there are almost certainly other cellular molecules/co-factors that connect/complex RIG-I and MDA-5 signaling to IPS-1, FADD and RIP-1 to mediate anti-viral innate immune responses. Accordingly, using a yeast two-hybrid screen for FADD-interacting proteins, a novel DexD/H box RNA helicase, Fah-1 (for Fadd-associated helicase) has been identified. Importantly, our analysis has confirmed that Fah-1 is required for cellular defense against virus infection. We therefore hypothesize that Fah-1 may participate in FADD-dependent innate immune responses, and propose the following Specific Aims to understand the role of Fah-1 in anti-viral host defense. 1.) Characterization of Fah-1 and molecular mechanisms of action. In this Aim, we outline experiments designed to further molecularly characterize Fah-1 and propose structure-function analyses to elucidate its interactions with FADD and/or other proteins involved in FADD-dependent innate immune signaling events. 2.) Role of Fah-1 in anti-viral innate immune responses. Our preliminary data indicate a critical role for Fah-1 in cellular anti-viral host defense. In this Aim, we will attempt to elucidate the role of Fah-1 in type I IFN induction and in anti-viral TLR-dependent and TLR-independent innate immunity. We anticipate that these studies will contribute towards understanding the molecular mechanisms of innate immune responses to virus infection. This information may also provide important and novel information on modes of viral-induced pathogenesis and importantly, will provide new concepts for the rational design of novel drugs and vaccine strategies. This project intends to elucidate mechanisms of how the cell recognizes and responds to virus infection. We anticipate that these studies will improve our knowledge of pathogenesis and provide new concepts for the rational design of novel therapeutic and vaccine strategies. [unreadable] [unreadable] [unreadable]